This Article Abstract Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Dimmeler, S. Articles by Zeiher, A. M. Search for Related Content PubMed PubMed Citation Articles by Dimmeler, S. Articles by Zeiher, A. M. Pubmed/NCBI databases Compound via MeSH Substance via MeSH Hazardous Substances DB NITRIC OXIDE

(Circulation Research. 1997;81:970-976.)
© 1997 American Heart Association, Inc.

Articles

Angiotensin II Induces Apoptosis of Human Endothelial Cells

Protective Effect of Nitric Oxide

Stefanie Dimmeler, Volker Rippmann, Ulrike Weiland, Judith Haendeler, , Andreas M. Zeiher

From Molecular Cardiology, Department of Internal Medicine IV, University of Frankfurt (Germany).

Correspondence to Stefanie Dimmeler, PhD, Department of Internal Medicine IV, Division of Cardiology, University of Frankfurt, Theodor-Stern-Kai 7, 60590 Frankfurt, Germany. E-mail Dimmeler{at}em.uni-frankfurt.de

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Abstract Angiotensin II (Ang II) importantly contributes to the pathobiology of atherosclerosis. Since endothelial injury is a key event early in the pathogenesis of atherosclerosis, we tested the hypothesis that Ang II may injure endothelial cells by activation of cellular suicide pathways leading to apoptosis. Human umbilical venous endothelial cells (HUVECs) were incubated with increasing doses of Ang II for 18 hours. Apoptosis of HUVECs was measured by ELISA specific for histone-associated DNA fragments and confirmed by DNA laddering and nuclear staining. Ang II dose-dependently induced apoptosis of HUVECs. Simultaneous blockade of both the AT₁ and AT₂ receptor prevented Ang II–induced apoptosis, whereas each individual receptor blocker alone was not effective. Selective agonistic stimulation of the AT₂ receptor also dose-dependently induced apoptosis. Ang II–mediated as well as selective AT₂ receptor stimulation–mediated apoptosis was associated with the activation of caspase-3, a central downstream effector of the caspase cascade executing the cell death program. Specific inhibition of caspase-3 activity abrogated Ang II–induced apoptosis. In addition, the NO donors sodium nitroprusside and S-nitrosopenicillamine completely inhibited Ang II–induced apoptosis and eliminated caspase-3 activity. Thus, Ang II induces apoptosis of HUVECs via activation of the caspase cascade, the central downstream effector arm executing the cell death program. NO completely abrogated Ang II–induced apoptosis by interfering with the activation of the caspase cascade.

Key Words: CPP32 • protease • CGP42112 • PD123177 • EXP3174

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Angiotensin II importantly contributes to the pathobiology of atherosclerosis and vascular disease not only via its role in hypertension but also via its direct effects on vascular cell growth and migration.^1,2 Ang II has previously been shown to promote the growth of VSMCs via activation of several growth factors like fibroblast growth factor and platelet-derived growth factor.² These growth-promoting effects of Ang II for VSMCs appear to be mediated by the AT₁ receptor.³ However, recent data indicate that Ang II represents a bifunctional growth factor by simultaneously stimulating proliferative and antiproliferative pathways.⁴ Indeed, in endothelial cells, Ang II mediates an antigrowth effect via stimulation of the AT₂ receptor.⁵ The mechanisms underlying the antimitogenic action of Ang II in endothelial cells are unknown.

Cellular proliferation is in large part determined by the balance between cell division and cell death by apoptosis. Apoptosis refers to the morphological alterations exhibited by "actively" dying cells that include cell shrinkage, membrane blebbing, chromatin condensation, and DNA fragmentation.⁶ The effector arm of the signal transduction pathway executing the cell death program is composed of cysteine proteases belonging to the ICE/CPP32 family, which have been recently termed caspases.⁷ Caspase-3, also referred to as CPP32/Yama,^8,9 has been shown to play an important role as a downstream member of the protease cascade, where various cell death pathways converge into the same effector pathway.¹⁰ On activation of the protease cascade, the caspase-3 proenzyme is proteolytically cleaved into the p17 and p12 subunits, which then heterodimerize to form the active enzyme.⁸

We have previously demonstrated that caspase-3 is activated in both the TNF-–mediated and growth factor withdrawal–induced apoptotic signal transduction pathways in human endothelial cells.^11,12 Therefore, the present study was designed to investigate whether the growth inhibitory effects of Ang II on HUVECs involve activation of the caspase cascade suggestive of the execution of the cell death program. In addition, since previous studies have consistently shown a countervailing balance between Ang II and NO with respect to the effects of these factors in the regulation of vessel tone and VSMC growth,^13,14 we examined any potentially antagonistic effects of NO on Ang II–induced apoptosis of HUVECs.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Reagents
HUVECs, endothelial basal medium, and supplements were purchased from Cell Systems/Clonetics, and FCS was from GIBCO. [³²P]dCTP was obtained from Amersham Klenow polymerase and cell death detection assay were from Boehringer Mannheim. PD123177, caspase-3-inhibitor, and substrate were obtained from Biomol. Ang II was from Sigma, and CGP42112 was from Neosystem Laboratoire. The antibody against CPP32(p20) was from Santa Cruz (clone D027). The guanylate cyclase inhibitor NS2028¹⁵ was kindly donated by Dr R. Busse (Frankfurt, Germany).

Cell Culture
HUVECs were cultured in endothelial basal medium supplemented with hydrocortisone (1 µg/mL), bovine brain extract (12 µg/mL), gentamicin (50 µg/mL), amphotericin B (50 ng/mL), epidermal growth factor (10 ng/mL), and 10% fetal calf serum until the third passage. After detachment with trypsin, cells were grown for at least 18 hours. All experiments were performed in the presence of complete medium including 10% fetal calf serum. Shear exposure was performed as previously outlined.^11,16 The rat smooth muscle cell line A-10 (DSMZ, Braunschweig, Germany) or human vascular smooth muscle cells were cultivated in DMEM with 20% fetal calf serum and 1% penicillamine and streptomycin. Before the experiment, the smooth muscle cells were starved for 48 hours in the absence of fetal calf serum.

DNA Fragmentation
DNA fragmentation analysis was carried out as recently described.^11,12 Cells were scraped off the plates and centrifuged at 700g for 10 minutes, washed with PBS, and resuspended in incubation buffer. The histone-associated DNA fragments were linked to the anti-histone antibody from mouse, and the DNA part of the nucleosome was linked to the anti–DNA-peroxidase. The amount of peroxidase retained in the immunocomplex was determined photometrically.

DNA Isolation and Klenow Labeling
Cells (1x10⁶), including detached cells, were removed from the culture flask and collected by centrifugation (10 minutes at 700g), washed with PBS, and incubated in lysis buffer (5 mmol/L Tris-HCl, pH 8, 20 mmol/L EDTA, and 0.5% Triton X-100) for 15 minutes at 4°C. After centrifugation for 20 minutes at 20 000g at 4°C, the supernatants were treated with RNase A for 1 hour at 37°C. A final concentration of 0.5 mg/mL proteinase K and 1% SDS was added, and the samples were incubated overnight at 65°C. After isolation of DNA by phenol-chloroform extraction, the DNA was precipitated with 70% isopropanol and 0.1 mol/L NaCl. The resulting pellet was resolved in TE buffer (10 mmol/L Tris-HCl, pH 8, and 1 mmol/L EDTA), and the DNA samples were incubated with 5 U Klenow polymerase and 0.5 µCi [³²P]dCTP according to Rösl.¹⁷ The reaction was terminated by the addition of 10 mmol/L EDTA, and the unincorporated nucleotides were removed by Sephadex G-50 columns. Labeled DNA fragments were separated on a 1.8% agarose gel, transferred to nitrocellulose membranes, and exposed to x-ray film.

Determination of Cell Viability
Cell viability was detected as described previously.¹⁸ HUVECs (1x10⁵ cells/mL) were grown in 96-well plates for 24 hours. After incubation with apoptotic stimuli for 18 hours, cells were treated with MTT (0.5 mg/mL) for 4 hours at 37°C. The cell culture medium was removed, cells were lysed in 2-isopropanol containing 0.04 mol/L HCl, and the amount of MTT was photometrically determined.

Fluorescence Staining
Cells were washed with PBS and fixed in 4% formaldehyde. Cells were stained with DAPI (0.2 µg/mL in 10 mmol/L Tris-HCl, pH 7, 10 mmol/L EDTA, and 100 mmol/L NaCl) for 30 minutes. Then cells were washed with PBS, and nuclei were analyzed by fluorescence microscopy. Two independent investigators counted apoptotic nuclei in a total cell number of 600 nuclei and expressed the result as apoptotic nuclei/600x100%.

Measurement of Caspase-3–Like Protease Activity
For detection of caspase-3–like activity, HUVECs (5x10⁵ cells) were lysed in buffer (1% Triton X-100, 0.32 mol/L sucrose, 5 mmol/L EDTA, 1 mmol/L phenylmethylsulfonyl fluoride, 1 µg/mL aprotinin, 1 µg/mL leupeptin, 2 mmol/L dithiothreitol, and 10 mmol/L Tris-HCl, pH 8) for 15 minutes at 4°C, followed by centrifugation (20 000g for 10 minutes). Caspase-3–like activity was detected in resulting supernatants by measuring the proteolytic cleavage of the fluorogenic substrate AMC-DEVD and AMC as standard in assay buffer containing 100 mmol/L HEPES, 10% sucrose, 0.1% CHAPS, pH 7.5, and 10 mmol/L dithiothreitol (excitation wavelength, 380 nm; emission wavelength, 460 nm).¹⁹ Enzyme activity was calculated as mol AMCxmg protein^-1xs^-1. Specificity for caspase-3–like enzymatic activity was demonstrated by inhibition with 10 nmol/L Ac-DEVD-CHO.^8,19 Protein content was analyzed using the Bio-Rad assay.

Western Blot
HUVECs were incubated, and protein was prepared as described for detection of caspase-3–like activity. Proteins (80 µg protein/slot) were resolved on 15% SDS-polyacrylamide gels and were blotted on nitrocellulose membranes by means of a semidry blotting system (3 mA/cm² for 30 minutes; buffer consisted of 48 mmol/L Tris, 39 mmol/L glycine, 0.037% SDS, and 20% methanol). The membranes were washed twice with TBS (50 mmol/L Tris-HCl, pH 8, 150 mmol/L NaCl, and 2.5 mmol/L KCl), and unspecific binding was blocked overnight at 4°C with 3% BSA in TBS/0.1% Tween 20. The antibody against the p17 subunit of human caspase-3 was added in a final dilution of 1:200 in TBS/3% BSA/0.1% Tween 20 for 1 hour at room temperature. After it was washed three times with TBS/Tween 20, the horseradish peroxidase–conjugated anti-goat IgG antibody (1:2000 in TBS/3% BSA/0.1% Tween 20) was incubated for 1 hour, and enhanced chemiluminescence was performed according to the instructions of the manufacturer.

Statistics
Statistical analysis was performed with ANOVA followed by a modified least significant difference (Bonferroni) test (SPSS-Software).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Ang II Induces Apoptosis of HUVECs
Incubation of HUVECs with Ang II dose-dependently induced apoptosis as determined by an ELISA specific for histone-associated DNA fragments (Fig 1a). Maximal DNA fragmentation was obtained after incubation for 18 hours, whereas prolonged incubation for 24 hours did not lead to a further increase of DNA fragmentation (data not shown). Ang II–induced apoptosis was confirmed by demonstration of the typical DNA-laddering pattern (Fig 1b) as well as by visual analysis of DAPI-stained cells (Fig 1c). Quantification after fluorescence staining revealed apoptosis in 4.5±1.7% of the cells after treatment with Ang II versus apoptosis in 1.7±2% of the control cells (n=4, P<.05). Furthermore, the appearance of DNA fragments correlated with a decrease of cell viability to 83±6% at 1 µmol/L Ang II compared with control cells. Necrosis was excluded by determination of the LDH release, which was not affected by Ang II treatment (105±5% apoptosis after 18-hour incubation with 1 µmol/L Ang II compared with control cells). In contrast to the induction of apoptosis in HUVECs, Ang II did not render rat smooth muscle cells apoptotic under similar conditions (Fig 1a). In addition, human smooth muscle cells were also not affected by 1 µmol/L Ang II (102±9% apoptosis compared with control cells).

View larger version (44K): [in this window] [in a new window]   Figure 1. Induction of apoptosis in HUVECs by Ang II or the AT2 receptor agonist CGP42112. a, Dose-dependent induction of DNA fragmentation by Ang II in HUVECs and lack of an effect on VSMCs after incubation for 18 hours as determined by ELISA (mean±SEM, n=4, *P<.05 vs 0.01 µmol/L Ang II). b, Representative DNA laddering induced in HUVECs by incubation of Ang II (1 µmol/L) for 18 hours. The top of the panel shows an ethidium bromide staining to ensure equal loading. c, DAPI fluorescence staining of nuclei after incubation in the presence or absence of Ang II (1 µmol/L) or CGP42112 (2 µmol/L) for 18 hours. Apoptotic cells are indicated by arrows. d, Influence of the AT1 and AT2 receptor antagonists, EXP3174 (Exp, 10 µmol/L) and PD123177 (PD, 10 µmol/L), respectively, on Ang II (1 µmol/L)–induced DNA fragmentation as assessed by ELISA after incubation for 18 hours (mean±SEM, n=4, *P<.05). e, Dose-dependent induction of apoptosis by the AT2 agonist CGP42112 in HUVECs after 18 hours of incubation (mean±SEM, n=4, *P<.05 vs 0.1 µmol/L CGP42112).

To characterize the angiotensin receptor subtype involved in the stimulation of apoptosis in HUVECs, we examined the influence of AT₁ and AT₂ receptor antagonists on Ang II–induced apoptosis. Ang II (1 µmol/L)–triggered apoptosis was not significantly affected by the specific AT₁ and AT₂ receptor antagonists, EXP3174 (1 and 10 µmol/L) and PD123177 (1 and 10 µmol/L), respectively (Fig 1d). However, simultaneous blockade of both AT₁ and AT₂ receptors by the combination of EXP3174 and PD123177 completely prevented Ang II–mediated apoptosis (Fig 1d). Similar effects were observed by stimulating apoptosis with 0.1 µmol/L Ang II (data not shown). Neither of the two antagonists alone nor their combination affected DNA fragmentation in the absence of Ang II (data not shown). Control experiments further ensured that the solvent ethanol did not influence apoptosis and viability of endothelial cells (DNA fragmentation, 212±68% of control in the presence of Ang II).

In order to further investigate a potential involvement of AT₂ receptor activation in the stimulation of apoptosis signal transduction, we additionally investigated the effects of the specific AT₂ receptor agonist CGP42112.⁵ As illustrated in Fig 1e, CGP42112 dose-dependently induced apoptosis.

To rule out the possibility that Ang II processing into shorter peptides²⁰ accounts for the proapoptotic effect of Ang II, we tested the Ang II peptides Ang(1–7) and Ang(3–8). Neither of the peptides induced apoptosis when incubated for 18 hours at a concentration of 1 µmol/L [Ang(1–7), 92±27% of control; Ang(3–8), 108±19% of control; n=3].

To finally ensure that AT₁ and AT₂ receptors are expressed in the HUVECs under study, AT₁ and AT₂ mRNA was detected by means of reverse-transcriptase PCR. Specific mRNA transcripts for both receptor subtypes were demonstrated in HUVECs by PCR analysis (oligonucleotides: AT₁, 5'-gccctgtccacaatatcttgc/5'-tgtaagattgcttcagccagc⁵; AT₂, 5'-cttcatttaatagctgtatgat/5'-ttgtggtttaaatacaaagca), which exhibited the predicted sizes (AT₁, 507 bp; AT₂, 590 bp).

Involvement of Caspase-3–Like Protease
The caspase proteases play a key role in apoptotic processes in HUVECs.^12,21 Therefore, the effect of Ang II on caspase-3–like activity was investigated. Ang II as well as the specific AT₂ receptor agonist CGP42112 activated caspase-3–like activity to 134±9% and 146±25%, respectively, compared with unstimulated cells. In addition, CGP42112 stimulated the proteolytical cleavage of caspase-3 into its active subunits, p12 and p17, as assessed by Western blot using an antibody that prevalently reacts with the active p17 subunit (Fig 2). Most important, the specific peptide caspase-3 inhibitor Ac-DEVD-CHO completely inhibited Ang II–stimulated apoptosis (Fig 3a), documenting the involvement of caspase-3–like enzymes in Ang II–induced DNA fragmentation. In addition, the Ang II–induced decrease of cell viability (83±6% of control) was completely prevented by 100 µmol/L Ac-DEVD-CHO (102±7% of control).

View larger version (29K): [in this window] [in a new window]   Figure 2. Effect of CGP42112 on caspase-3 cleavage into its active subunits. Western blot against caspase-3 and the active subunit p17 of HUVEC homogenates after treatment with or without NO donors (SNP, 10 µmol/L; SNAP, 10 µmol/L) for 1 hour and subsequent stimulation with CGP42112 (2 µmol/L) for 18 hours.

View larger version (22K): [in this window] [in a new window]   Figure 3. Effect of NO, specific caspase-3 inhibitor, and 8-bromo-cGMP on Ang II–induced apoptosis and caspase-3–like activity. a, Inhibition of Ang II (1 µmol/L)–induced apoptosis by SNP (10 µmol/L), SNAP (10 µmol/L), Ac-DEVD-CHO (100 µmol/L), or 8-bromo-cGMP (1 mmol/L) after incubation for 18 hours (mean±SEM, n=4). b, Influence of the AT1 or AT2 receptor antagonists EXP3174 (Exp, 10 µmol/L) and PD123177 (PD, 10 µmol/L) in combination with SNAP (10 µmol/L) on Ang II (1 µmol/L)–induced apoptosis (mean±SEM, *P<.05). c, Effect of preincubation of SNP (10 µmol/L), SNAP (10 µmol/L), or 8-bromo-cGMP (1 mmol/L) for 1 hour on CGP42112 (CGP, 2 µmol/L; 18-hour incubation)–induced DNA fragmentation (mean±SEM, *P<.05). d, Caspase-3–like-activity determined after incubation of CGP (2 µmol/L) for 18 hours. SNP (10 µmol/L) or SNAP (10 µmol/L) was preincubated for 1 hour (mean±SEM, n=3).

NO Inhibits Ang II–Induced Apoptosis
Since we have previously shown that NO inhibits caspase-3–like activity in HUVECs,¹² we investigated the effects of the NO donors SNP and SNAP. SNP (10 µmol/L) as well as SNAP (10 µmol/L) significantly reduced Ang II–induced apoptosis (Fig 3a). Inhibition of Ang II–induced apoptosis by NO was observed in the presence of AT₁ or AT₂ receptor blockade (Fig 3b), suggesting that the inhibitory effect of NO was due to interference with AT₁ and AT₂ receptor–mediated signal transduction. In addition, the specific stimulation of AT₂ receptor–induced DNA fragmentation with CGP42112 was completely prevented by the NO donors SNP and SNAP (Fig 3c). To determine the effect of endogenous NO, endothelial cells were exposed to laminar shear stress for 6 hours, which led to an increase of endothelial NO synthase protein levels (data not shown and References 12 and 16^{12 16} ). Preexposure of shear stress for 6 hours completely prevented further induction of apoptosis by Ang II (94±18% after preexposure to shear stress compared with 189±46%).

Inhibition of Ang II–induced apoptosis by NO appeared to be cGMP independent, since the cGMP analogue 8-bromo-cGMP did not affect Ang II–induced or CGP42112-induced DNA fragmentation (Fig 3a and 3c, respectively). In addition, the guanylate cyclase inhibitor NS2028¹⁵ did not affect the inhibitory effect of NO on Ang II–induced apoptosis (113±24% of control in the presence of 1 µmol/L Ang II, 10 µmol/L SNP, and 1 µmol/L NS2028; P<.05 versus Ang II). In order to elucidate the mechanism underlying the protective effect of NO, we investigated the influence on caspase-3–like activity and cleavage. SNP and SNAP abrogated the AT₂ receptor–triggered caspase-3–like activity and additionally prevented CGP42112-induced cleavage into the active subunits (Fig 2 and 3d).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The results of the present study demonstrate that Ang II induces apoptosis of human endothelial cells via activation of the caspase cascade, as evidenced by an increase of caspase-3–like enzyme activity and proteolytical cleavage of caspase-3 into its active subunits, p12 and p17. Furthermore, inhibition of caspase-3–like activity completely abrogated Ang II–induced apoptosis of HUVECs, indicating that activation of the caspase cascade plays a central role in executing the Ang II–stimulated cell death program. Importantly, NO antagonized the effects of Ang II to induce apoptosis of HUVECs, further supporting the concept of the countervailing influences of NO and Ang II in vascular biology. These findings considerable extend the role of Ang II as a potentially important contributor to the pathobiology of atherosclerosis.

The growth-promoting effects of Ang II have generally been attributed to stimulation of the AT₁ receptor.³ However, recent studies in nonendothelial cells have demonstrated that Ang II is also capable of inducing apoptosis via stimulation of the AT₂ receptor,²² which is primarily expressed during ontogenesis.²³ Moreover, it has been suggested that the antigrowth effect of Ang II on endothelial cells is mediated by the AT₂ receptor,⁵ although the underlying mechanisms have not been elucidated thus far. The results of the present study for the first time demonstrate that Ang II induces the execution of the cell death program in HUVECs. Endothelial cells have been shown to express both AT₁ and AT₂ receptor subtypes.⁵ Indeed, stimulation of the AT₂ receptor by high concentrations of the AT₂-selective analogue CGP42112 in the present study not only induced DNA fragmentation (indicative of the induction of apoptosis) but also significantly increased caspase-3–like activity and led to the proteolytical cleavage of caspase-3. Thus, activation of the cell death program by Ang II clearly involves stimulation of the AT₂ receptor in HUVECs. However, blocking the AT₂ receptor did not abolish the proapoptotic effects of Ang II, suggesting that Ang II–induced apoptosis of HUVECs is not exclusively mediated by the AT₂ receptor but depends on the complex interplay between both the AT₁ and AT₂ receptor, because simultaneously blocking both receptors eliminated Ang II–induced apoptosis. Given the pleiotropic AT₁ receptor–mediated effects of Ang II on a variety of second-messenger systems, such as protein kinase C, calcium, mitogen-activated kinases, and other tyrosine kinases, which all have been implicated in the regulation of cellular survival signals,^24,25 it is not surprising that blocking of individual receptors did not result in a complete reversal of the effects of Ang II. In addition, it is still an open debate whether more than two Ang II receptors are present in endothelial cells with as-yet-unknown functions and, probably more important, unknown affinities for the receptor blockers currently used. In addition, shorter fragments of Ang II were identified in human plasma and in tissues with distinct effects mediated via non-AT₁ and non-AT₂ receptors.²⁰ However, the processed Ang II fragments Ang(1–7) and Ang(3–8) did not affect endothelial cell apoptosis, suggesting that the proapoptotic effect of Ang II is not mediated via its metabolites. Regardless of the specific type of Ang II receptor responsible for the proapoptotic effects of Ang II, the results of the present study unambiguously demonstrate that the divergent signaling pathways coupled to the AT₁ and AT₂ receptor types converge into the activation of the caspase cascade, executing the cell death program in HUVECs on stimulation with Ang II.

The intracellular signal transduction mechanisms following Ang II receptor stimulation leading to activation of the caspase cascade in HUVECs remain to be determined. In nonendothelial cells, Ang II has been shown to dephosphorylate MAP kinase via AT₂-mediated activation of MAP kinase phosphatase,²² thereby counteracting the effects of growth-stimulatory signals. Whether such a mechanism is operative in endothelial cells is unknown at present. Moreover, there are no data providing support for a link between dephosphorylation of MAP kinases and activation of the caspase cascade. Ang II has been shown to induce the production of oxygen radicals, namely, superoxide anion, in a variety of cells, including endothelial cells.²⁶ The generation of reactive oxygen species has been demonstrated to be involved in mediating apoptosis via activation of the cell death program and stimulation of caspase activity.^27,28 However, Ang II–induced oxidative stress is mediated via activation of the AT₁ receptor in human endothelial cells and, therefore, most likely cannot account for the activation of the caspase cascade by the selective AT₂ receptor agonist observed in the present study.

NO donors completely abrogated Ang II–induced apoptosis in HUVECs and prevented cleavage of caspase-3. NO was equipotent in inhibiting Ang II–induced apoptosis compared with the effects of the specific tetrapeptide aldehyde inhibitor of caspase-3. These findings not only underscore the pivotal role of the caspase cascade as the downstream effector arm executing the Ang II–induced cell death program in HUVECs but also point toward the potent effects of NO to inhibit this effector pathway of apoptosis. Indeed, we have previously shown that NO inhibited TNF-–triggered apoptosis in HUVECs via S-nitrosylation of the reactive cysteine group within the active center of caspase-3 and caspase-1, thereby abrogating enzyme activity.¹² The results of the present study considerably extend these findings by demonstrating that NO-mediated inhibition of the caspase cascade exerts an important physiological function, namely, antagonizing the proapoptotic effects of Ang II in endothelial cells. Thus, the present study provides another example for the countervailing effects of Ang II and NO in vascular biology.

The results of the present study are apparently contradictory to recently published data by Pollman et al,¹⁴ who demonstrated that Ang II exerts antiapoptotic effects and directly antagonized NO-induced apoptosis in cultured VSMCs. However, these apparent discrepancies are easily reconciled. First, cultured VSMCs exclusively express AT₁ and no AT₂ receptors²⁹; second, the doses of NO used to induce apoptosis of VSMCs in the study by Pollman et al exceeded 50 µmol/L. It is well known that high doses of NO induce apoptosis via a direct DNA-damaging mechanism,³⁰ whereas low concentrations of NO have been shown to be protective against apoptosis.^12,31–33 Thus, the findings of the present study illustrate that it is extremely important not only to interpret the effects of Ang II in a cell type–specific manner but also to recognize the role of NO as a bifunctional modulator of cell fate capable of either inhibiting or stimulating cell death, depending on the concentration of NO applied. Nevertheless, since the endothelial NO synthase is known to produce small amounts of NO,³⁴ the countervailing autocrine effects between NO and Ang II in endothelial cells in vivo will most likely be inhibition of Ang II–induced apoptosis by NO.

The demonstration that Ang II induces apoptosis of endothelial cells may have important clinical implications by extending the potential mechanisms involved in the well-established role of Ang II to be a major contributor to the pathobiology of atherosclerosis.³⁵ Whereas previous studies have mainly focused on the effect of Ang II to promote growth and migration of VSMCs as a key feature of atherosclerotic lesion development,³⁶ the present study demonstrates that Ang II causes an injurious insult leading to apoptotic death of endothelial cells. Lesion-prone regions are characterized by an increased endothelial cell turnover rate,³⁷ which most likely is due to an increase of apoptotic cell death, suggesting a mechanistic link between endothelial cell turnover and the susceptibility to atherosclerotic plaque development.³⁸ Indeed, we have recently shown that oxidized low density lipoprotein, which is a well-established triggering molecule in the atherosclerotic process, also induces apoptosis of HUVECs.²¹ Taken together, the demonstration that Ang II induces apoptosis of endothelial cells may provide a mechanistic clue linking activity of the renin-angiotensin system with the "response-to-injury" hypothesis of atherogenesis. Activation of the renin-angiotensin system has been repeatedly shown to be associated with the presence of coronary atherosclerosis, especially in patients without additional risk factors.^39,40

In summary, the results of the present study demonstrate that Ang II induces apoptosis of human endothelial cells via activation of the caspase cascade, the central downstream effector arm executing the cell death program in response to a variety of stimuli. In accordance with the countervailing effects of Ang II and NO in the regulation of vessel tone and cell growth, NO completely abrogated Ang II–induced apoptosis of HUVECs by interfering with the activation of the caspase cascade. Thus, NO protects endothelial cells from being driven into cell death by Ang II. These findings have important implications not only with respect to the countervailing balance of NO and Ang II to determine vascular lesion formation but may also provide a mechanistic clue linking Ang II with the "response-to-injury" hypothesis of atherogenesis.

	Selected Abbreviations and Acronyms

 

Ac-DEVD-CHO = acetylated DEVD-CHO AMC = 7-amino-4-coumarin Ang II = angiotensin II CHO = Chinese hamster ovary DAPI = 4',6-diamidino-2-phenylindole DEVD = Asp-Glu-Val-Asp MAP kinase = mitogen-activated protein kinase MTT = 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide PCR = polymerase chain reaction SNAP = S-nitrosopenicillamine SNP = sodium nitroprusside VSMC = vascular smooth muscle cell

	Acknowledgments

 
This study was supported by grants from the Deutsche Forschungsgemeinschaft Di 600/2–2. Dr Dimmeler has a fellowship from the Deutsche Forschungsgemeinschaft. We would like to thank Christine Goebel for expert technical assistance.

Received June 26, 1997; accepted September 25, 1997.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 
1. Dzau VJ. Multiple pathways of angiotensin production in the blood vessel wall: evidence, possibilities and hypotheses. J Hypertens. 1989;7:933–936.[Medline] [Order article via Infotrieve]

2. Itoh H, Mukoyama M, Pratt RE, Gibbons GH, Dzau VJ. Multiple autocrine growth factors modulate vascular smooth muscle cell growth response to angiotensin II. J Clin Invest. 1993;91:2268–2274.

3. Lyall F, Dornan ES, McQueen J, Boswell F, Kelly M. Angiotensin II increases proto-oncogene expression and phosphoinositide turnover in vascular smooth muscle cells via the angiotensin II AT1 receptor. J Hypertens. 1992;10:1463–1469.[Medline] [Order article via Infotrieve]

4. Dzau VJ, Gibbons GH, Pratt RE. Molecular mechanisms of vascular renin-angiotensin system in myointimal hyperplasia. Hypertension. 1991;18(suppl II):II-100-II-105.

5. Stoll M, Steckelings UM, Paul M, Bottari SP, Metzger R, Unger T. The angiotensin AT2-receptor mediates inhibition of cell proliferation in coronary endothelial cells. J Clin Invest. 1995;95:651–657.

6. White E. Life, death, and the pursuit of apoptosis. Genes Dev. 1996;10:1–15.[Free Full Text]

7. Alnemri ES, Livingston DJ, Nicholson DW, Salvesen G, Wong WW, Yuan J. Human ICE/CED-3 protease nomenclature. Cell. 1996;87:171.[Medline] [Order article via Infotrieve]

8. Nicholson DW, Ali A, Thornberry NA, Vaillancourt JP, Ding CK, Gallant M, Gareau Y, Griffin PR, Labelle M, Lazebnik YA, et al. Identification and inhibition of the ICE/CED-3 protease necessary for mammalian apoptosis. Nature. 1995;376:37–43.[Medline] [Order article via Infotrieve]

9. Tewari M, Quan LT, O'Rourke K, Desnoyers S, Zeng Z, Beidler DR, Poirier GG, Salvesen GS, Dixit VM. Yama/CPP32 beta, a mammalian homolog of CED-3, is a CrmA-inhibitable protease that cleaves the death substrate poly(ADP-ribose) polymerase. Cell. 1995;81:801–809.[Medline] [Order article via Infotrieve]

10. Kumar S. ICE-like proteases in apoptosis. Trends Biochem Sci. 1995;20:198–202.[Medline] [Order article via Infotrieve]

11. Dimmeler S, Haendeler J, Rippmann V, Nehls M, Zeiher AM. Shear stress inhibits apoptosis of human endothelial cells. FEBS Lett. 1996;399:71–74.[Medline] [Order article via Infotrieve]

12. Dimmeler S, Haendeler J, Nehls M, Zeiher AM. Suppression of apoptosis by nitric oxide via inhibition of ICE-like and CPP32-like proteases. J Exp Med. 1997;185:601–608.[Abstract/Free Full Text]

13. Gibbons GH. Mechanisms of vascular remodeling in hypertension: role of autocrine-paracrine vasoactive factors. Curr Opin Nephrol Hypertens. 1995;4:189–196.[Medline] [Order article via Infotrieve]

14. Pollman MJ, Yamada T, Horiuchi M, Gibbons GH. Vasoactive substances regulate vascular smooth muscle cell apoptosis: countervailing influences of nitric oxide and angiotensin II. Circ Res. 1996;79:748–756.[Abstract/Free Full Text]

15. Mülsch A, Bauersachs J, Schäfer A, Stasch J-P, Kast R, Busse R. Effect of YC-1, an NO-independent, superoxide-sensitive stimulator of soluble guanylyl cyclase, on smooth muscle responsiveness to nitrovasodilators. Br J Pharmacol. 1997;120:681–689.[Medline] [Order article via Infotrieve]

16. Noris M, Morigi M, Donadelli R, Aiello S, Foppolo M, Todeschini M, Orisio S, Remuzzi G, Remuzzi A. Nitric oxide synthesis by cultured endothelial cells is modulated by flow conditions. Circ Res. 1995;76:536–543.[Abstract/Free Full Text]

17. Rösl F. A simple and rapid method for detection of apoptosis in human cells. Nucleic Acids Res. 1992;20:5243.[Free Full Text]

18. Dimmeler S, Brinkmann S, Neugebauer E. Endotoxin-induced changes of endothelial cell viability and permeability: protective effect of a 21-aminosteroid. Eur J Pharmacol. 1995;287:257–261.[Medline] [Order article via Infotrieve]

19. Bump NJ, Hackett M, Hugunin M, Seshagiri S, Brady K, Chen P, Ferenz C, Franklin S, Ghayur T, Li P, Licari P, Mankovich J, Shi L, Greenberg AH, Miller LK, Wong WW. Inhibition of ICE family proteases by baculovirus antiapoptotic protein p35. Science. 1995;269:1885–1888.[Abstract/Free Full Text]

20. Tallant EA, Lu X, Weiss RB, Chappell MC, Ferrario CM. Bovine aortic endothelial cells contain an angiotensin-(1–7) receptor. Hypertension. 1997;29:388–393.[Abstract/Free Full Text]

21. Dimmeler S, Haendeler J, Galle J, Zeiher AM. Oxidized low density lipoprotein induces apoptosis of human endothelial cells by activation of CPP32-like proteases: a mechanistic clue to the response to injury hypothesis. Circulation. 1997;95:1760–1763.[Abstract/Free Full Text]

22. Yamada T, Horiuchi M, Dzau VJ. Angiotensin II type 2 receptor mediates programmed cell death. Proc Natl Acad Sci U S A. 1996;93:156–160.[Abstract/Free Full Text]

23. Grady EF, Sechi LA, Griffin CA, Schambelan M, Kalinyak JE. Expression of AT2 receptors in the developing rat fetus. J Clin Invest. 1991;88:921–933.

24. Griendling KK, Alexander RW. The angiotensin (AT1) receptor. Semin Nephrol. 1993;13:558–566.[Medline] [Order article via Infotrieve]

25. Liao D-F, Duff JL, Daum G, Pelech SL, Berk BC. Angiotensin II stimulates MAP kinase kinase kinase activity in vascular smooth muscle cells: role of Raf. Circ Res. 1996;79:1007–1014.[Abstract/Free Full Text]

26. Oskarsson HJ, Heistad DD. Oxidative stress produced by angiotensin too: implications for hypertension and vascular disease. Circulation. 1997;95:557–559.[Free Full Text]

27. Jacobson MD. Reactive oxygen species and programmed cell death. Trends Biochem Sci. 1996;21:83–86.[Medline] [Order article via Infotrieve]

28. Troy CM, Stefanis L, Prochiantz A, Greene LA, Shelanski ML. The contrasting roles of ICE family proteases and interleukin-1ß in apoptosis induced by trophic factor withdrawal and by copper/zinc superoxide dismutase down regulation. Proc Natl Acad Sci U S A. 1996;93:5635–5640.[Abstract/Free Full Text]

29. De Gasparo M, Whitebread S, Mele M, Montani AS, Whitecombe PJ, Ramjoue HP, Kamber B. Biochemical characterization of two angiotensin II receptor subtypes in the rat. J Cardiovasc Pharmacol. 1990;16(suppl):S31–S35.

30. Nguyen T, Brunson D, Crespi CL, Penman BW, Wishnok JS, Tannenbaum SR. DNA damage and mutation in human cells exposed to nitric oxide in vitro. Proc Natl Acad Sci U S A. 1992;89:3030–3034.[Abstract/Free Full Text]

31. Mannick JB, Asano K, Izumi K, Kieff E, Stamler JS. Nitric oxide produced by human B lymphocytes inhibits apoptosis and Epstein-Barr virus reactivation. Cell. 1994;79:1137–1146.[Medline] [Order article via Infotrieve]

32. Genaro AM, Hortelano S, Alvarez A, Martinez C, Bosca L. Splenic B lymphocyte programmed cell death is prevented by nitric oxide release through mechanisms involving sustained Bcl-2 levels. J Clin Invest. 1995;95:1884–1890.

33. Kim Y-M, de Vera ME, Watkins SC, Billiar TR. Nitric oxide protects cultured rat hepatocytes from tumor necrosis factor-alpha-induced apoptosis by inducting heat shock protein 70 expression. J Biol Chem. 1997;272:1402–1411.[Abstract/Free Full Text]

34. Lefer DJ, Scalia R, Campbell B, Nossuli T, Hayward R, Salamon M, Grayson J. Peroxynitrite inhibits leukocyte-endothelial cell interactions and protects against ischemia-reperfusion injury in rats. J Clin Invest. 1997;99:684–691.[Medline] [Order article via Infotrieve]

35. Chobanian AV, Dzau VJ. Renin angiotensin system and atherosclerotic vascular disease. In: Fuster V, Ross R, Topol EJ, eds. Atherosclersosis and Coronary Artery Disease. Philadelphia, Pa: Lippincott-Raven; 1996;1:237–243.

36. Ross R. Cell biology of atherosclerosis. Annu Rev Physiol. 1995;57:791–804.[Medline] [Order article via Infotrieve]

37. Caplan BA, Schwartz CJ. Increased endothelial cell turnover in areas of in vivo Evans blue uptake in the pig aorta. Atherosclerosis. 1973;17:401–417.[Medline] [Order article via Infotrieve]

38. Ross R. The pathogenesis of atherosclerosis: an update. N Engl J Med. 1986;314:488–500.[Medline] [Order article via Infotrieve]

39. Cambien F, Poirier O, Lecerf L, Evans A, Cambou JP, Arveiler D, Luc G, Bard JM, Bara L, Ricard S, et al. Deletion polymorphism in the gene for angiotensin-converting enzyme is a potent risk factor for myocardial infarction. Nature. 1992;359:641–644.[Medline] [Order article via Infotrieve]

40. Alderman MH, Madhavan S, Ooi WL, Cohen H, Sealey JE, Laragh JH. Association of the renin-sodium profile with the risk of myocardial infarction in patients with hypertension. N Engl J Med. 1991;324:1098–1104.[Abstract]

This article has been cited by other articles:

				A. M. Briones, N. Rodriguez-Criado, R. Hernanz, A. B. Garcia-Redondo, R. R. Rodrigues-Diez, M. J. Alonso, J. Egido, M. Ruiz-Ortega, and M. Salaices Atorvastatin Prevents Angiotensin II-Induced Vascular Remodeling and Oxidative Stress Hypertension, July 1, 2009; 54(1): 142 - 149. [Abstract] [Full Text] [PDF]

				S. Reungjui, C. A. Roncal, W. Sato, O. Y. Glushakova, B. P. Croker, S.-i. Suga, X. Ouyang, K. Tungsanga, T. Nakagawa, R. J. Johnson, et al. Hypokalemic Nephropathy is Associated with Impaired Angiogenesis J. Am. Soc. Nephrol., January 1, 2008; 19(1): 125 - 134. [Abstract] [Full Text] [PDF]

				J.-S. Jerng, Y.-C. Hsu, H.-D. Wu, H.-Z. Pan, H.-C. Wang, C.-T. Shun, C.-J. Yu, and P.-C. Yang Role of the renin-angiotensin system in ventilator-induced lung injury: an in vivo study in a rat model Thorax, June 1, 2007; 62(6): 527 - 535. [Abstract] [Full Text] [PDF]

				N. Chitravas, H. M. Dewey, M. B. Nicol, D. L. Harding, D. C. Pearce, and A. G. Thrift Is prestroke use of angiotensin-converting enzyme inhibitors associated with better outcome? Neurology, May 15, 2007; 68(20): 1687 - 1693. [Abstract] [Full Text] [PDF]

				P. C. Joshi and D. M. Guidot The alcoholic lung: epidemiology, pathophysiology, and potential therapies Am J Physiol Lung Cell Mol Physiol, April 1, 2007; 292(4): L813 - L823. [Abstract] [Full Text] [PDF]

				M. El Chaar, J. Chen, S. V. Seshan, S. Jha, I. Richardson, S. R. Ledbetter, E. D. Vaughan Jr, D. P. Poppas, and D. Felsen Effect of combination therapy with enalapril and the TGF-beta antagonist 1D11 in unilateral ureteral obstruction Am J Physiol Renal Physiol, April 1, 2007; 292(4): F1291 - F1301. [Abstract] [Full Text] [PDF]

				N. J. Meyer and J. G. N. Garcia Wading into the Genomic Pool to Unravel Acute Lung Injury Genetics Proceedings of the ATS, January 1, 2007; 4(1): 69 - 76. [Abstract] [Full Text] [PDF]

				T. A. Williams, A. Verhovez, A. Milan, F. Veglio, and P. Mulatero Protective Effect of Spironolactone on Endothelial Cell Apoptosis Endocrinology, May 1, 2006; 147(5): 2496 - 2505. [Abstract] [Full Text] [PDF]

				M. Sata Role of Circulating Vascular Progenitors in Angiogenesis, Vascular Healing, and Pulmonary Hypertension: Lessons From Animal Models Arterioscler Thromb Vasc Biol, May 1, 2006; 26(5): 1008 - 1014. [Abstract] [Full Text] [PDF]

				P. Chanvorachote, U. Nimmannit, L. Wang, C. Stehlik, B. Lu, N. Azad, and Y. Rojanasakul Nitric Oxide Negatively Regulates Fas CD95-induced Apoptosis through Inhibition of Ubiquitin-Proteasome-mediated Degradation of FLICE Inhibitory Protein J. Biol. Chem., December 23, 2005; 280(51): 42044 - 42050. [Abstract] [Full Text] [PDF]

				M. Akishita, K. Nagai, H. Xi, W. Yu, N. Sudoh, T. Watanabe, M. Ohara-Imaizumi, S. Nagamatsu, K. Kozaki, M. Horiuchi, et al. Renin-Angiotensin System Modulates Oxidative Stress-Induced Endothelial Cell Apoptosis in Rats Hypertension, June 1, 2005; 45(6): 1188 - 1193. [Abstract] [Full Text] [PDF]

				T. Watanabe, T. A. Barker, and B. C. Berk Angiotensin II and the Endothelium: Diverse Signals and Effects Hypertension, February 1, 2005; 45(2): 163 - 169. [Abstract] [Full Text] [PDF]

				C. J. Wruck, H. Funke-Kaiser, T. Pufe, H. Kusserow, M. Menk, J. H. Schefe, M. L. Kruse, M. Stoll, and T. Unger Regulation of Transport of the Angiotensin AT2 Receptor by a Novel Membrane-Associated Golgi Protein Arterioscler Thromb Vasc Biol, January 1, 2005; 25(1): 57 - 64. [Abstract] [Full Text] [PDF]

				S. I. Sokol, E. L. Portnay, J. P. Curtis, M. A. Nelson, P. R. Hebert, J. F. Setaro, and J. M. Foody Modulation of the renin-angiotensin-aldosterone system for the secondary prevention of stroke Neurology, July 27, 2004; 63(2): 208 - 213. [Abstract] [Full Text] [PDF]

				B. Chandrasekar, K. Vemula, R. M. Surabhi, M. Li-Weber, L. B. Owen-Schaub, L. E. Jensen, and S. Mummidi Activation of Intrinsic and Extrinsic Proapoptotic Signaling Pathways in Interleukin-18-mediated Human Cardiac Endothelial Cell Death J. Biol. Chem., May 7, 2004; 279(19): 20221 - 20233. [Abstract] [Full Text] [PDF]

				N. Tejera, D. Gomez-Garre, A. Lazaro, J. Gallego-Delgado, C. Alonso, J. Blanco, A. Ortiz, and J. Egido Persistent Proteinuria Up-Regulates Angiotensin II Type 2 Receptor and Induces Apoptosis in Proximal Tubular Cells Am. J. Pathol., May 1, 2004; 164(5): 1817 - 1826. [Abstract] [Full Text] [PDF]

				H. Ohashi, H. Takagi, H. Oh, K. Suzuma, I. Suzuma, N. Miyamoto, A. Uemura, D. Watanabe, T. Murakami, T. Sugaya, et al. Phosphatidylinositol 3-Kinase/Akt Regulates Angiotensin II-Induced Inhibition of Apoptosis in Microvascular Endothelial Cells by Governing Survivin Expression and Suppression of Caspase-3 Activity Circ. Res., April 2, 2004; 94(6): 785 - 793. [Abstract] [Full Text] [PDF]

				G. Wolf and U. O. Wenzel Angiotensin II and Cell Cycle Regulation Hypertension, April 1, 2004; 43(4): 693 - 698. [Abstract] [Full Text] [PDF]

				X. Li, H. Rayford, and B. D. Uhal Essential Roles for Angiotensin Receptor AT1a in Bleomycin-Induced Apoptosis and Lung Fibrosis in Mice Am. J. Pathol., December 1, 2003; 163(6): 2523 - 2530. [Abstract] [Full Text]

				R. Benndorf, R. H. Boger, S. Ergun, A. Steenpass, and T. Wieland Angiotensin II Type 2 Receptor Inhibits Vascular Endothelial Growth Factor-Induced Migration and In Vitro Tube Formation of Human Endothelial Cells Circ. Res., September 5, 2003; 93(5): 438 - 447. [Abstract] [Full Text] [PDF]

				E.-L. Marchand, S. Der Sarkissian, P. Hamet, and D. deBlois Caspase-Dependent Cell Death Mediates the Early Phase of Aortic Hypertrophy Regression in Losartan-Treated Spontaneously Hypertensive Rats Circ. Res., April 18, 2003; 92(7): 777 - 784. [Abstract] [Full Text] [PDF]

				M. Weis and J. P. Cooke Cardiac Allograft Vasculopathy and Dysregulation of the NO Synthase Pathway Arterioscler Thromb Vasc Biol, April 1, 2003; 23(4): 567 - 575. [Abstract] [Full Text] [PDF]

				Y. Zhang, J. M. Lehman, and L. M. Petti Apoptosis of Mortal Human Fibroblasts Transformed by the Bovine Papillomavirus E5 Oncoprotein Mol. Cancer Res., December 1, 2002; 1(2): 122 - 136. [Abstract] [Full Text] [PDF]

				J. Agata, L. Chao, and J. Chao Kallikrein Gene Delivery Improves Cardiac Reserve and Attenuates Remodeling After Myocardial Infarction Hypertension, November 1, 2002; 40(5): 653 - 659. [Abstract] [Full Text] [PDF]

				Y.-J. Geng and P. Libby Progression of Atheroma: A Struggle Between Death and Procreation Arterioscler Thromb Vasc Biol, September 1, 2002; 22(9): 1370 - 1380. [Abstract] [Full Text] [PDF]

				J. Suzuki, M. Iwai, H. Nakagami, L. Wu, R. Chen, T. Sugaya, M. Hamada, K. Hiwada, and M. Horiuchi Role of Angiotensin II-Regulated Apoptosis Through Distinct AT1 and AT2 Receptors in Neointimal Formation Circulation, August 13, 2002; 106(7): 847 - 853. [Abstract] [Full Text] [PDF]

				G. Ding, K. Reddy, A. A. Kapasi, N. Franki, N. Gibbons, B. S. Kasinath, and P. C. Singhal Angiotensin II induces apoptosis in rat glomerular epithelial cells Am J Physiol Renal Physiol, July 1, 2002; 283(1): F173 - F180. [Abstract] [Full Text] [PDF]

				M. Papp, X. Li, J. Zhuang, R. Wang, and B. D. Uhal Angiotensin receptor subtype AT1 mediates alveolar epithelial cell apoptosis in response to ANG II Am J Physiol Lung Cell Mol Physiol, April 1, 2002; 282(4): L713 - L718. [Abstract] [Full Text] [PDF]

				F. J. Miller Jr, W. J. Sharp, X. Fang, L. W. Oberley, T. D. Oberley, and N. L. Weintraub Oxidative Stress in Human Abdominal Aortic Aneurysms: A Potential Mediator of Aneurysmal Remodeling Arterioscler Thromb Vasc Biol, April 1, 2002; 22(4): 560 - 565. [Abstract] [Full Text] [PDF]

				M. Stoll, A. W.A. Hahn, U. Jonas, Y. Zhao, B. Schieffer, J. W. Fischer, and T. Unger Identification of a Zinc Finger Homoeodomain Enhancer Protein After AT2 Receptor Stimulation by Differential mRNA Display Arterioscler Thromb Vasc Biol, February 1, 2002; 22(2): 231 - 237. [Abstract] [Full Text] [PDF]

				C. Berry, R. Touyz, A. F. Dominiczak, R. C. Webb, and D. G. Johns Angiotensin receptors: signaling, vascular pathophysiology, and interactions with ceramide Am J Physiol Heart Circ Physiol, December 1, 2001; 281(6): H2337 - H2365. [Abstract] [Full Text] [PDF]

				M. Ruiz-Ortega, O. Lorenzo, M. Ruperez, V. Esteban, Y. Suzuki, S. Mezzano, J.J. Plaza, and J. Egido Role of the Renin-Angiotensin System in Vascular Diseases: Expanding the Field Hypertension, December 1, 2001; 38(6): 1382 - 1387. [Abstract] [Full Text] [PDF]

				G. S. Filippatos, N. Gangopadhyay, O. Lalude, N. Parameswaran, S. I. Said, W. Spielman, and B. D. Uhal Regulation of apoptosis by vasoactive peptides Am J Physiol Lung Cell Mol Physiol, October 1, 2001; 281(4): L749 - L761. [Abstract] [Full Text] [PDF]

				J. W Fischer, M. Stoll, A. W.A Hahn, and T. Unger Differential regulation of thrombospondin-1 and fibronectin by angiotensin II receptor subtypes in cultured endothelial cells Cardiovasc Res, September 1, 2001; 51(4): 784 - 791. [Abstract] [Full Text] [PDF]

				B. C. Berk Vascular Smooth Muscle Growth: Autocrine Growth Mechanisms Physiol Rev, July 1, 2001; 81(3): 999 - 1030. [Abstract] [Full Text] [PDF]

				D.-H. KANG, A. H. JOLY, S.-W. OH, C. HUGO, D. KERJASCHKI, K. L. GORDON, M. MAZZALI, J. A. JEFFERSON, J. HUGHES, K. M. MADSEN, et al. Impaired Angiogenesis in the Remnant Kidney Model: I. Potential Role of Vascular Endothelial Growth Factor and Thrombospondin-1 J. Am. Soc. Nephrol., July 1, 2001; 12(7): 1434 - 1447. [Abstract] [Full Text] [PDF]

				J. Culman, J. Baulmann, A. Blume, and T. Unger Review: The renin-angiotensin system in the brain: an update Journal of Renin-Angiotensin-Aldosterone System, June 1, 2001; 2(2): 96 - 102. [PDF]

				Y.-p. Sun, B.-q. Zhu, A. E. M. Browne, S. Pulukurthy, T. M. Chou, K. Sudhir, S. A. Glantz, P. C. Deedwania, K. Chatterjee, and W. W. Parmley Comparative Effects of ACE Inhibitors and an Angiotensin Receptor Blocker on Atherosclerosis and Vascular Function Journal of Cardiovascular Pharmacology and Therapeutics, June 1, 2001; 6(2): 175 - 181. [Abstract] [PDF]

				H. Sugino, R. Ozono, S. Kurisu, H. Matsuura, M. Ishida, T. Oshima, M. Kambe, Y. Teranishi, H. Masaki, and H. Matsubara Apoptosis Is Not Increased in Myocardium Overexpressing Type 2 Angiotensin II Receptor in Transgenic Mice Hypertension, June 1, 2001; 37(6): 1394 - 1398. [Abstract] [Full Text] [PDF]

				Z. Mallat and A. Tedgui Current Perspective on the Role of Apoptosis in Atherothrombotic Disease Circ. Res., May 25, 2001; 88(10): 998 - 1003. [Abstract] [Full Text] [PDF]

				D.-H. Kang, Y.-G. Kim, T. F. Andoh, K. L. Gordon, S.-I. Suga, M. Mazzali, J. A. Jefferson, J. Hughes, W. Bennett, G. F. Schreiner, et al. Post-cyclosporine-mediated hypertension and nephropathy: amelioration by vascular endothelial growth factor Am J Physiol Renal Physiol, April 1, 2001; 280(4): F727 - F736. [Abstract] [Full Text] [PDF]

				M.-S. Zhou, A. Adam, and L. Raij Review: Interaction among angiotensin II, nitric oxide and oxidative stress Journal of Renin-Angiotensin-Aldosterone System, March 1, 2001; 2(1_suppl): S59 - S63. [PDF]

				J. L Mehta and Dayuan Li Facilitative interaction between angiotensin II and oxidised LDL in cultured human coronary artery endothelial cells Journal of Renin-Angiotensin-Aldosterone System, March 1, 2001; 2(1_suppl): S70 - S76. [Abstract] [PDF]

				T.-X. Cui, H. Nakagami, M. Iwai, Y. Takeda, T. Shiuchi, L. Daviet, C. Nahmias, and M. Horiuchi Pivotal role of tyrosine phosphatase SHP-1 in AT2 receptor-mediated apoptosis in rat fetal vascular smooth muscle cell Cardiovasc Res, March 1, 2001; 49(4): 863 - 871. [Abstract] [Full Text] [PDF]

				L. Rossig, J. Haendeler, Z. Mallat, B. Hugel, J.-M. Freyssinet, A. Tedgui, S. Dimmeler, and A. M. Zeiher Congestive heart failure induces endothelial cell apoptosis: protective role of carvedilol J. Am. Coll. Cardiol., December 1, 2000; 36(7): 2081 - 2089. [Abstract] [Full Text] [PDF]

				R. M. Touyz and E. L. Schiffrin Signal Transduction Mechanisms Mediating the Physiological and Pathophysiological Actions of Angiotensin II in Vascular Smooth Muscle Cells Pharmacol. Rev., December 1, 2000; 52(4): 639 - 672. [Abstract] [Full Text] [PDF]

				S. W. Rabkin and J. Y Kong Nitroprusside induces cardiomyocyte death: interaction with hydrogen peroxide Am J Physiol Heart Circ Physiol, December 1, 2000; 279(6): H3089 - H3100. [Abstract] [Full Text] [PDF]

				Z.-G. Jin, M. G. Melaragno, D.-F. Liao, C. Yan, J. Haendeler, Y.-A. Suh, J. D. Lambeth, and B. C. Berk Cyclophilin A Is a Secreted Growth Factor Induced by Oxidative Stress Circ. Res., October 27, 2000; 87(9): 789 - 796. [Abstract] [Full Text] [PDF]

				D. Li, T. Saldeen, F. Romeo, and J. L. Mehta Oxidized LDL Upregulates Angiotensin II Type 1 Receptor Expression in Cultured Human Coronary Artery Endothelial Cells : The Potential Role of Transcription Factor NF-{kappa}B Circulation, October 17, 2000; 102(16): 1970 - 1976. [Abstract] [Full Text] [PDF]

				E. O. Harrington, A. Smeglin, N. Parks, J. Newton, and S. Rounds Adenosine induces endothelial apoptosis by activating protein tyrosine phosphatase: a possible role of p38alpha Am J Physiol Lung Cell Mol Physiol, October 1, 2000; 279(4): L733 - L742. [Abstract] [Full Text] [PDF]

				J. L. Hall, C. M. Matter, X. Wang, and G. H. Gibbons Hyperglycemia Inhibits Vascular Smooth Muscle Cell Apoptosis Through a Protein Kinase C-Dependent Pathway Circ. Res., September 29, 2000; 87(7): 574 - 580. [Abstract] [Full Text] [PDF]

				M. de Gasparo, K. J. Catt, T. Inagami, J. W. Wright, and Th. Unger International Union of Pharmacology. XXIII. The Angiotensin II Receptors Pharmacol. Rev., September 1, 2000; 52(3): 415 - 472. [Abstract] [Full Text] [PDF]

				K. Irani Oxidant Signaling in Vascular Cell Growth, Death, and Survival : A Review of the Roles of Reactive Oxygen Species in Smooth Muscle and Endothelial Cell Mitogenic and Apoptotic Signaling Circ. Res., August 4, 2000; 87(3): 179 - 183. [Abstract] [Full Text] [PDF]

				J. Sadoshima Cytokine Actions of Angiotensin II Circ. Res., June 23, 2000; 86(12): 1187 - 1189. [Full Text] [PDF]

				N. Elbaz, K. Bedecs, M. Masson, M. Sutren, A. D. Strosberg, and C. Nahmias Functional Trans-inactivation of Insulin Receptor Kinase by Growth-Inhibitory Angiotensin II AT2 Receptor Mol. Endocrinol., June 1, 2000; 14(6): 795 - 804. [Abstract] [Full Text]

				G. H. Gibbons and M. J. Pollman Death Receptors, Intimal Disease, and Gene Therapy : Are Therapies That Modify Cell Fate Moving too Fas? Circ. Res., May 26, 2000; 86(10): 1009 - 1012. [Full Text] [PDF]

				J. Hughes, M. Nangaku, C. E. Alpers, S. J. Shankland, W. G. Couser, and R. J. Johnson C5b-9 membrane attack complex mediates endothelial cell apoptosis in experimental glomerulonephritis Am J Physiol Renal Physiol, May 1, 2000; 278(5): F747 - F757. [Abstract] [Full Text] [PDF]

				J. Haendeler, M. Ishida, L. Hunyady, and B. C. Berk The Third Cytoplasmic Loop of the Angiotensin II Type 1 Receptor Exerts Differential Effects on Extracellular Signal-Regulated Kinase (ERK1/ERK2) and Apoptosis via Ras- and Rap1-Dependent Pathways Circ. Res., April 14, 2000; 86(7): 729 - 736. [Abstract] [Full Text] [PDF]

				C Berry and A.L Clark Catabolism in chronic heart failure Eur. Heart J., April 1, 2000; 21(7): 521 - 532. [PDF]

				D. Lang, S. I. Mosfer, A. Shakesby, F. Donaldson, and M. J. Lewis Coronary Microvascular Endothelial Cell Redox State in Left Ventricular Hypertrophy : The Role of Angiotensin II Circ. Res., March 3, 2000; 86(4): 463 - 469. [Abstract] [Full Text] [PDF]

				S. Gallinat, S. Busche, M. K. Raizada, and C. Sumners The angiotensin II type 2 receptor: an enigma with multiple variations Am J Physiol Endocrinol Metab, March 1, 2000; 278(3): E357 - E374. [Abstract] [Full Text] [PDF]

				M. Horiuchi, W. Hayashida, M. Akishita, S. Yamada, J. Y. A. Lehtonen, K. Tamura, L. Daviet, Y. E. Chen, M. Hamai, T.-X. Cui, et al. Interferon-{gamma} Induces AT2 Receptor Expression in Fibroblasts by Jak/STAT Pathway and Interferon Regulatory Factor-1 Circ. Res., February 4, 2000; 86(2): 233 - 240. [Abstract] [Full Text] [PDF]

				D. Lang, M. B. Kredan, S. J. Moat, S. A. Hussain, C. A. Powell, M. F. Bellamy, H. J. Powers, and M. J. Lewis Homocysteine-Induced Inhibition of Endothelium-Dependent Relaxation in Rabbit Aorta : Role for Superoxide Anions Arterioscler Thromb Vasc Biol, February 1, 2000; 20(2): 422 - 427. [Abstract] [Full Text] [PDF]

				S. B. Parker, A. D. Dobrian, S. S. Wade, and R. L. Prewitt AT1 receptor inhibition does not reduce arterial wall hypertrophy or PDGF-A expression in renal hypertension Am J Physiol Heart Circ Physiol, February 1, 2000; 278(2): H613 - H622. [Abstract] [Full Text] [PDF]

				M. M Kockx and A. G Herman Apoptosis in atherosclerosis: beneficial or detrimental? Cardiovasc Res, February 1, 2000; 45(3): 736 - 746. [Abstract] [Full Text] [PDF]

				M. AKISHITA, M. HORIUCHI, H. YAMADA, L. ZHANG, G. SHIRAKAMI, K. TAMURA, Y. OUCHI, and V. J. DZAU Inflammation influences vascular remodeling through AT2 receptor expression and signaling Physiol Genomics, January 24, 2000; 2(1): 13 - 20. [Abstract] [Full Text] [PDF]

				S. D. Kim Measurement of the Renin-Angiotensin System in Heart Failure Biol Res Nurs, January 1, 2000; 1(3): 210 - 226. [Abstract] [PDF]

				R. P. Mason Calcium channel blockers, apoptosis and cancer: is there a biologic relationship? J. Am. Coll. Cardiol., December 1, 1999; 34(7): 1857 - 1866. [Abstract] [Full Text] [PDF]

				E. Chamoux Involvement of the Angiotensin II Type 2 Receptor in Apoptosis during Human Fetal Adrenal Gland Development J. Clin. Endocrinol. Metab., December 1, 1999; 84(12): 4722 - 4730. [Abstract] [Full Text]

				R. Wang, C. Ramos, I. Joshi, A. Zagariya, A. Pardo, M. Selman, and B. D. Uhal Human lung myofibroblast-derived inducers of alveolar epithelial apoptosis identified as angiotensin peptides Am J Physiol Lung Cell Mol Physiol, December 1, 1999; 277(6): L1158 - L1164. [Abstract] [Full Text] [PDF]

				Q. N. Diep, J.-S. Li, and E. L. Schiffrin In Vivo Study of AT1 and AT2 Angiotensin Receptors in Apoptosis in Rat Blood Vessels Hypertension, October 1, 1999; 34(4): 617 - 624. [Abstract] [Full Text] [PDF]

				N. Wang, L. Verna, S. Hardy, Y. Zhu, K.-S. Ma, M. J. Birrer, and M. B. Stemerman c-Jun Triggers Apoptosis in Human Vascular Endothelial Cells Circ. Res., September 3, 1999; 85(5): 387 - 393. [Abstract] [Full Text] [PDF]

				T. Stefanec Circulating Apoptotic Endothelial Cells Blood, August 15, 1999; 94(4): 1482 - 1483. [Full Text] [PDF]

				J. Y. A. Lehtonen, L. Daviet, C. Nahmias, M. Horiuchi, and V. J. Dzau Analysis of Functional Domains of Angiotensin II Type 2 Receptor Involved in Apoptosis Mol. Endocrinol., July 1, 1999; 13(7): 1051 - 1060. [Abstract] [Full Text]

				J.-E. Fabre, A. Rivard, M. Magner, M. Silver, and J. M. Isner Tissue Inhibition of Angiotensin-Converting Enzyme Activity Stimulates Angiogenesis In Vivo Circulation, June 15, 1999; 99(23): 3043 - 3049. [Abstract] [Full Text] [PDF]

				D. Y. Li, Y. C. Zhang, M. I. Philips, T. Sawamura, and J. L. Mehta Upregulation of Endothelial Receptor for Oxidized Low-Density Lipoprotein (LOX-1) in Cultured Human Coronary Artery Endothelial Cells by Angiotensin II Type 1 Receptor Activation Circ. Res., May 14, 1999; 84(9): 1043 - 1049. [Abstract] [Full Text] [PDF]

				R. Wang, A. Zagariya, O. Ibarra-Sunga, C. Gidea, E. Ang, S. Deshmukh, G. Chaudhary, J. Baraboutis, G. Filippatos, and B. D. Uhal Angiotensin II induces apoptosis in human and rat alveolar epithelial cells Am J Physiol Lung Cell Mol Physiol, May 1, 1999; 276(5): L885 - L889. [Abstract] [Full Text] [PDF]

				M. Horiuchi, W. Hayashida, M. Akishita, K. Tamura, L. Daviet, J. Y. A. Lehtonen, and V. J. Dzau Stimulation of Different Subtypes of Angiotensin II Receptors, AT1 and AT2 Receptors, Regulates STAT Activation by Negative Crosstalk Circ. Res., April 30, 1999; 84(8): 876 - 882. [Abstract] [Full Text] [PDF]

				M. Horiuchi, M. Akishita, and V. J. Dzau Recent Progress in Angiotensin II Type 2 Receptor Research in the Cardiovascular System Hypertension, February 1, 1999; 33(2): 613 - 621. [Abstract] [Full Text] [PDF]

				D. J. Ing, J. Zang, V. J. Dzau, K. A. Webster, and N. H. Bishopric Modulation of Cytokine-Induced Cardiac Myocyte Apoptosis by Nitric Oxide, Bak, and Bcl-x Circ. Res., January 22, 1999; 84(1): 21 - 33. [Abstract] [Full Text] [PDF]

				U. V. Shenoy, E. M. Richards, X.-C. Huang, and C. Sumners Angiotensin II Type 2 Receptor-Mediated Apoptosis of Cultured Neurons from Newborn Rat Brain Endocrinology, January 1, 1999; 140(1): 500 - 509. [Abstract] [Full Text]

				P. J. M. Best, D. Hasdai, G. Sangiorgi, R. S. Schwartz, D. R. Holmes Jr, R. D. Simari, and A. Lerman Apoptosis : Basic Concepts and Implications in Coronary Artery Disease Arterioscler Thromb Vasc Biol, January 1, 1999; 19(1): 14 - 22. [Abstract] [Full Text] [PDF]

				M. Horiuchi, H. Yamada, M. Akishita, M. Ito, K. Tamura, and V. J. Dzau Interferon Regulatory Factors Regulate Interleukin-1ߖConverting Enzyme Expression and Apoptosis in Vascular Smooth Muscle Cells Hypertension, January 1, 1999; 33(1): 162 - 166. [Abstract] [Full Text] [PDF]

				J.-D. Chiche, S. M. Schlutsmeyer, D. B. Bloch, S. M. de la Monte, J. D. Roberts Jr., G. Filippov, S. P. Janssens, A. Rosenzweig, and K. D. Bloch Adenovirus-mediated Gene Transfer of cGMP-dependent Protein Kinase Increases the Sensitivity of Cultured Vascular Smooth Muscle Cells to the Antiproliferative and Pro-apoptotic Effects of Nitric Oxide/cGMP J. Biol. Chem., December 18, 1998; 273(51): 34263 - 34271. [Abstract] [Full Text] [PDF]

				A. M. Diehl Roles of CCAAT/Enhancer-binding Proteins in Regulation of Liver Regenerative Growth J. Biol. Chem., November 20, 1998; 273(47): 30843 - 30846. [Abstract] [Full Text] [PDF]

				D. H. Walter, J. Haendeler, J. Galle, A. M. Zeiher, and S. Dimmeler Cyclosporin A Inhibits Apoptosis of Human Endothelial Cells by Preventing Release of Cytochrome C From Mitochondria Circulation, September 22, 1998; 98(12): 1153 - 1157. [Abstract] [Full Text] [PDF]

				S. Kotamraju, N. Hogg, J. Joseph, L. K. Keefer, and B. Kalyanaraman Inhibition of Oxidized Low-density Lipoprotein-induced Apoptosis in Endothelial Cells by Nitric Oxide. PEROXYL RADICAL SCAVENGING AS AN ANTIAPOPTOTIC MECHANISM J. Biol. Chem., May 11, 2001; 276(20): 17316 - 17323. [Abstract] [Full Text] [PDF]

				U. Schmitz, K. Thommes, I. Beier, W. Wagner, A. Sachinidis, R. Dusing, and H. Vetter Angiotensin II-induced Stimulation of p21-activated Kinase and c-Jun NH2-terminal Kinase Is Mediated by Rac1 and Nck J. Biol. Chem., June 15, 2001; 276(25): 22003 - 22010. [Abstract] [Full Text] [PDF]

				J.-R. Nofer, B. Levkau, I. Wolinska, R. Junker, M. Fobker, A. von Eckardstein, U. Seedorf, and G. Assmann Suppression of Endothelial Cell Apoptosis by High Density Lipoproteins (HDL) and HDL-associated Lysosphingolipids J. Biol. Chem., September 7, 2001; 276(37): 34480 - 34485. [Abstract] [Full Text] [PDF]

				J. Hoffmann, J. Haendeler, A. Aicher, L. Rossig, M. Vasa, A. M. Zeiher, and S. Dimmeler Aging Enhances the Sensitivity of Endothelial Cells Toward Apoptotic Stimuli: Important Role of Nitric Oxide Circ. Res., October 12, 2001; 89(8): 709 - 715. [Abstract] [Full Text] [PDF]

				F. J. Miller Jr, W. J. Sharp, X. Fang, L. W. Oberley, T. D. Oberley, and N. L. Weintraub Oxidative Stress in Human Abdominal Aortic Aneurysms: A Potential Mediator of Aneurysmal Remodeling Arterioscler Thromb Vasc Biol, April 1, 2002; 22(4): 560 - 565. [Abstract] [Full Text] [PDF]

This Article Abstract Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Dimmeler, S. Articles by Zeiher, A. M. Search for Related Content PubMed PubMed Citation Articles by Dimmeler, S. Articles by Zeiher, A. M. Pubmed/NCBI databases Compound via MeSH Substance via MeSH Hazardous Substances DB NITRIC OXIDE